Abstract
Single-molecule technologies can provide detailed information regarding molecular mechanisms and interactions that cannot easily be studied on the bulk scale; generally, individual molecular behaviors cannot be distinguished, and only average characteristics can be measured. Nevertheless, the development of the single-molecule sequencer had a significant impact on conventional in vitro single-molecule research, featuring automated equipment, high-throughput chips, and automated analysis systems. However, the utilization of sequencing technology in in vitro single-molecule research is not yet globally prevalent, owing to the large gap between highly organized single-molecule sequencing and manual-based in vitro single-molecule research. Here, we describe the principles of zero-mode waveguides (ZMWs) and nanopore methods used as single-molecule DNA sequencing techniques, and provide examples of functional biological measurements beyond DNA sequencing that contribute to a global understanding of the current applications of these sequencing technologies. Furthermore, through a comparison of these two technologies, we discuss future applications of DNA sequencing technologies in in vitro single-molecule research.